Toluidine blue staining of murine mast cells and quantitation by a novel, automated image analysis method using whole slide skin images.

Mast cells are immune cells of myeloid lineage, characterized by the presence of cytoplasmic granules. These cells play a significant role in multiple pathologies, such as urticaria and type 1 hypersensitivity reactions. Mast cells in tissue sections can be demonstrated with metachromatic stains, such as toluidine blue. Metachromatic staining of mast cells is influenced by the type of mast cell, pH of the staining solution and fixative employed. The objective of this study is to identify a simple, consistent, and reproducible toluidine blue staining method to quantify the mast cells in whole slide skin images using automated image analysis. Skin sections from naive mice and atopic dermatitis mice were stained with toluidine blue methods by Churukian-Schenk and Luna. Luna's toluidine blue staining method without the eosin counterstaining provided optimal staining of mast cells with a greater contrast to the background. The Churukian-Schenk toluidine blue method resulted in slight background staining which confounded the accurate detection and quantification of mast cells in mouse skin sections using an automated image analysis algorithm. Using the Luna toluidine blue stain, a 5-fold increase in the total number of mast cells was observed in atopic dermatitis skin samples as compared to naive mice. In summary, a simple, conventional, and reproducible toluidine blue method was identified to quantify the mast cells in mouse skin sections using an automated image analysis algorithm.